The protist parasite Trypanosoma brucei causes lethal diseases in humans and livestock animals, and is transmitted by its tsetse vector. A key strategy in gene expression of this pathogen is to utilize highly active RNA polymerase (pol) I for the transcription of genes encoding variant surface glycoprotein (VSG) and procyclin. This is unique because in eukaryotes RNA pol I exclusively transcribe ribosomal DNA whereas all mRNA is synthesized by RNA pol II. The glycoproteins are essential for the parasite because they form a protective cell surface coat. Moreover, antigenic variation of the VSG coat in bloodstream form trypanosomes is the means by which the parasite evades the mammalian immune system. The multifunctional use of RNA pol I in T. brucei involves recruitment of the enzyme to four structurally different promoter types during the parasite's life cycle, concentration of the enzyme in two distinct nuclear compartments in bloodstream forms, and life cycle-dependent regulation of VSG and procyclin gene transcription. This functional diversity predicts that T. brucei RNA pol I undergoes essential interactions with a greater variety of factors or factor domains than its host counterparts. The long-term goal of this proposal is to understand the parasite-specific biology of RNA pol I transcription in T. brucei and to identify essential factors, factor domains, or protein-protein interactions which might be exploited for parasite control. The proposed study aims at: 1. Characterization and purification of RNA pol I complexes from both procyclic and bloodstream form trypanosomes. As in other organisms, RNA pol I may form a holoenzyme whose purification may include basal transcription factors and other transcription regulatory proteins. It will be of particular interest to identify a life cycle-specific component. 2. Functional characterization of parasite-specific transcription factors or factor domains. We have already identified such a domain at the N-terminus of the RNA pol I second largest subunit. 3. Extending the genetic analysis from promoters to transcription enhancer and termination elements. 4. Purification of our in vitro transcription activity to enable characterization of specific DNA-protein-interactions, which may facilitate purification and identification of auxiliary transcription factors.